The Golgi consists of a stack of flattened membrane compartments that is essential for the sequential processing and sorting of all secretory and transmembrane proteins before they are delivered to their final destinations. Yet a clear understanding of the role and regulation of Golgi stacking on the protein trafficking functions of the Golgi has remained elusive due to the lack of experimental methods to induce Golgi unstacking in vivo. For example, many studies have relied on investigating the disassembled Golgi from mitotic extracts that is limited to in vitro experiments. RNAi-mediated knockdown of potential stacking factors did not produce unstacked Golgi, perhaps due to compensatory mechanisms that kick-in during the long silencing period. The long-term objective of this project is to investigate the structure-function relationship of the Golgi by devising a new approach to rapidly unstack the Golgi in live cells. Then, its effect on Golgi function and dynamics will be directly visualized in real time using live cell imaging. The first aim will involve the constructon of a knock-sideways system for Golgi stacking factors GRASP65 and GRASP55. This will allow their drug-induced inactivation at the Golgi in cells where RNAi has silenced the endogenous counterpart. The unstacking of nocodazole-induced peripheral Golgi mini-stacks will be monitored by triple color total internal reflection fluorescence (TIRF) microscopy, which has the resolution needed to distinguish cis- and trans-cisternae. The second aim will set up a live cell imaging assay that can visualize intra-Golgi trafficking in real time. TIRF microscopy will be used to directly monitor cargo entry into the cis- or trans-cisternae in stacked and unstacked Golgi in order to test the hypothesis that Golgi stacking is mandatory for intra-Golgi trafficking. This work will provide important insight into the most conserved feature of Golgi membrane structure and function. In addition, a better understanding of the mechanism of Golgi misregulation in human genetic disorders and cancers related to membrane trafficking may be attained.